Abstract

Purpose: To localize specific components of the Basal Membrane
Complex (BMC) of elongating lens fibers at defined points in their
migration to the posterior sutures.

Methods: Normal, juvenile (4-6 week old) Sprague-Dawley rat lenses
(n=46) were utilized. Lenses were either decapsulated to obtain whole
mounts of lens capsules or sectioned with a vibrating knife microtome.
Sections (100 µm thick) were cut parallel to the equatorial plane,
beginning at the posterior pole. On both sections and whole mounts,
F-actin was localized using phalloidin-FITC while myosin, cadherins,
and β1 integrin were localized using immunofluorescent labeling.
Specimens were visualized on a laser scanning confocal microscope.

Results: F-actin labeling in the equatorial and peri-sutural regions
was predominately localized to the periphery of basal fiber ends
(consistent with our prior results). At sutures, labeling for F-actin
in the BMC was rearranged into numerous small profiles. Furthermore,
labeling intensity for F-actin was increased at sutures. Myosin was
present in the BMC in all locations examined as a diffuse plaque at
fiber ends. Similarly, β1 integrin was also distributed throughout the
BMC within the actin-rich borders in all regions except adjacent to and
at the suture branches. In that location immunofluorescence for β1
integrin appeared to be reduced. In the equatorial, lateral-posterior,
and peri-sutural regions, cadherin showed strong localization around
the periphery of basal fiber ends. However, cadherin labeling was
markedly reduced in the BMC as fibers detached from the capsule and
abutted to form sutures (i.e. in the sutural region). Cadherin was
concentrated along the short faces of elongating fiber mid-segments.

Conclusions: It appears that F-actin, cadherin and β1 integrin
components of the BMC undergo controlled rearrangements in the final
stages of migration and detachment from the capsule.

Elongation of nascent lens fibers involves the migration of basal
fiber ends along the lens capsule from the meridional rows at the
equator to their sutural destinations on the lens posterior. The
interaction of migrating basal fiber ends with the capsule is mediated
by the basal membrane complex (BMC). The BMC is defined as the fiber
cell basal membrane, its resident integral membrane proteins, and their
associated cytoskeletal and regulatory elements. In avian lenses, BMC
components include integrins, cadherins, actin, myosin, caldesmon,
paxillin, focal adhesion kinase (FAK), and myosin light chain kinase
(MLCK) [1].

Components of the BMC, such as actin, β1 integrin, and N-cadherin,
have been studied to elucidate their role in cell attachment to the
extra cellular matrix (ECM), proliferation, and migration. It is well
established that β1 integrins are the primary integrin receptor for the
basal lamina proteins of the lens capsule [2]. These integrins function as bidirectional
signaling receptors, mediating cytoskeletal-ECM interactions that
impact fiber adhesion and polarization [3]. The cell adhesion molecule, N-cadherin, also
plays a role in lens fiber differentiation, specifically regulating
differentiation-dependent cytoskeletal reorganization via its linkage
to actin [4].
Cadherins are also essential to the systematic formation and
organization of cell-cell interactions during migration [5]. It is therefore not
surprising that N-cadherin has been co-localized with actin both in the
native BMC and in cell culture [1,4].
Actin has been found to play a key role in coordinating structural
changes and maintaining the integrity of lens development during fiber
cell elongation [6-10].

During fiber migration, fiber ends must reach precise destinations,
detach from the capsule at the appropriate time, and overlap and/or
abut with opposing fiber ends. This convergence of opposing fiber ends
results in the formation of lens sutures. Although fibers differentiate
and elongate to form sutures in a similar manner in all vertebrate
lenses, their migration patterns are not identical, leading to
variations in sutural anatomy among species. Specifically, there are
four distinct suture patterns: branchless or umbilical sutures, line
sutures, Y sutures, and star sutures (see [11] for a detailed review). In avian and
reptile lenses, fiber ends migrate essentially straight toward the
poles where they detach from the capsule (posteriorly) or epithelium
(anteriorly) to overlap and abut. This growth scheme results in a
system in which all fibers are meridians, and branchless (umbilical)
sutures are formed.

In all other vertebrate lenses, elongating fiber ends within each
growth shell have diverse migration paths, resulting in fibers of
variable length and curvature, which overlap and abut to form branched
sutures. These lenses have two basic types of fibers, those with
straight end segments (straight fibers) and those with curved end
segments (curved fibers). Straight fibers extend to a pole on one end
and to the proximal end of a suture branch on the other end. Curved
fibers lie between the straight fibers. The simplest branched suture
pattern is formed when fiber ends (of curved fibers) migrate in one of
four directions, resulting in a line suture wherein the two branches
are oriented 180° apart. These are seen in rabbit and amphibian lenses.
Similarly, in lenses with Y sutures, fiber ends within a growth shell
migrate in one of six directions to form a three-branched, “Y” pattern.
Y sutures are found in rodent, feline, canine, porcine, ovine, and
bovine lenses. Finally, when fiber ends in each growth shell migrate in
one of twelve directions, a six-branched, simple star suture pattern
results. This simple star pattern is characteristic of primate lenses
and, in humans, becomes increasingly complex with age. Each of the four
suture patterns has a specific effect on lens optical quality as
measured by focal ability [12].

From the above, it can be seen that proper formation of lens sutures
is crucial in establishing and maintaining structural order (at the
cellular level), which minimizes large particle scatter thus promoting
transparency [13].
Furthermore, because defined suture patterns are only formed via
coordinated fiber end migration, it is clear that migration is a key
process in producing appropriate fiber organization. In addition to
maximizing lens clarity, structural order is known to impact lens
function. Specifically, a number of studies have demonstrated that
excessive disorganization of newly elongated fibers and lens suture
patterns results in a degradation of lens focal ability [14-16] and if extreme,
may compromise lens transparency [17,18].

While it is clear that both the anterior and posterior sutures exert
a negative effect on the transparency and refractive properties of
lenses, the process of posterior suture formation appears particularly
vulnerable to compromise leading to excessive disorder and eventual
cataract [19,20]. In fact, prior
structural analyses have suggested that faulty fiber migration wherein
the posterior sutures fail to form results in posterior subcapsular
cataract (PSC) formation in several animal models [21-26]. Thus, it is
essential to gain an understanding of all aspects of basal fiber end
migration including morphology, organization, migration patterns and
the identity and distribution of BMC components, in order to evaluate
how and why this process is disrupted in pathological situations.

Although the distribution of several BMC molecules was described in
fibers at the beginning of elongation [1], little is known about their molecular
arrangement during the terminal phases of migration and formation of
lens sutures. Our recent investigations have shown that the morphology
and organization of basal fiber ends in mammalian lenses displays
significant variation during migration, especially during the terminal
phases [27].
Additionally, BMC architecture in mammalian lenses, which feature
branched suture patterns, may be substantially different from that
reported for avian lenses, which have branchless, umbilical sutures.
Therefore, the goal of this study was to localize specific BMC
components in rodent elongating lens fibers at both the initial and
terminal stages of fiber end migration. Our results indicate that the
distribution of several BMC components alters as fiber ends approach
their sutural destinations.

Lenses

Normal Sprague-Dawley rats (4-8 weeks old) were utilized for this
investigation. All animals were handled in compliance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research and
with Rush University Medical Center IACUC (Institutional Animal Care
and Use Committee) guidelines. A total of 48 animals were sacrificed by
intraperitoneal injection of a euthanasia agent, and the eyes were
immediately enucleated.

Whole-mount lens capsules

Whole mounts of the posterior lens capsule were prepared as
previously described [27].
Briefly, lenses were pre-fixed for 2 min in 2% paraformaldehyde, washed
in 0.07 M phosphate buffer saline (PBS), and placed on dental wax with
the posterior surface down. The capsule was lightly grasped near the
anterior pole, then carefully peeled away from the lens and pinned to
the dental wax. The decapsulated fiber mass was gently removed and the
capsule (with the sheared-off fiber ends adhered) was fixed for 30 min
in 3% paraformaldehyde in 0.07 M (PBS). This procedure generally yields
an intact posterior lens capsule with attached basal fiber ends in the
initial regions of fiber end migration (defined previously [27]), specifically the
equatorial region and the proximal portion of the posterior lateral
region. Basal ends in more distal locations do not readily remain
adhered to the rat lens capsule, even after the pre-fixation procedure.

Vibratome sectioning

In order to examine the distribution of BMC components in fiber ends
distal to the equator, posterior polar sections were utilized. Sections
have the additional advantage of yielding intact fiber ends, whereas
the capsule stripping method may introduce mild artifacts in fiber end
morphology due to tension created during the stripping process (wherein
fiber ends are sheared off from the remainder of the cells).

Before sectioning, the posterior retina and sclera were removed and
the remainder of the eye was placed in 3% paraformaldehyde in 0.07 M
PBS. After fixation for 2 h, the lenses were dissected from the eye and
briefly rinsed in 0.07 M PBS. In order to obtain posterior sections,
lenses were mounted on a sectioning chuck, anterior side down, with
cyanoacrylate glue (Figure
1A) and embedded in warm 2% agar, which solidified at room
temperature. Lenses were sectioned parallel to the equator with a
vibrating knife microtome (Lancer Series 1000 or Vibratome 3000 Series;
Vibratome, St. Louis, MO). Sections 100 μm thick were cut beginning at
the posterior pole (Figure
1B) using an amplitude of 3 and a speed of 5 (approximately
0.6 mm/sec). Sections were fixed for an additional 30 min in 3%
paraformaldehyde, briefly washed in 0.07 M PBS, and then processed for
immunocytochemistry.

Ideally, the first vibratome section should yield the enveloping
posterior capsule, the posterior fiber ends of elongating fibers, and
the nascent suture branches, as well as the underlying growth shells of
cortical maturing fibers. Fiber “feet” should be seen in all locations
beneath the rounded, uncut posterior lens surface (Figure 1C; #1).
In subsequent sections, fiber feet should be present under the capsule
only beneath the beveled edge of the section (Figure 1C; #2). In some instances
sections were retrieved at or near the equator of the lens by
continuing to section deeper into the specimen.

Immunocytochemistry

Immunolabeling was performed by sequentially immersing sections in
drops of solution as follows. Sections were permeabilized in 0.2%
Triton X-100 for 30 min followed by a 1 h incubation in a blocking
solution of 10% goat serum (for N-cadherin, pan-cadherin and myosin IIA
labeling), 10% rabbit serum (for β-1 integrin labeling), or 10% Donkey
Serum (for actin I-19, myosin IIA, and pan-cadherin multiple labeling),
1% bovine serum albumin (BSA) and 0.05% thimerosal in 0.07 M PBS. The
sections were then reacted with a primary antibody specific to the BMC
component of interest (actin, β-1 integrin, N-cadherin, pan-cadherin,
or myosin IIA) for at least 2 h at room temperature or overnight at 4º
C. Primary antibodies from different host animals were combined into a
single solution during this step for multiple labeling experiments
(triple labeling). Appropriate primary antibody dilutions were
determined by dilution series tests; dilutions were made in the
blocking solution. Following three 10 min washes in blocking solution,
sections were incubated in a secondary antibody conjugated to a
fluorescent label (FITC, TRITC, AMCA, or a combination of each) for at
least 2 h at room temperature. Sections were washed 4 times in 0.07 M
PBS for 10 min each, then mounted on glass slides (Gold Seal Products,
Portsmouth, NH) with Vectashield mounting medium (Vector Laboratories,
Inc., Burlington, CA) to prevent photo bleaching. Specimens were
examined on either a Zeiss LSM 510 or Zeiss LSM 510 Meta laser scanning
confocal microscope (Research Resource Center, University of Illinois
at Chicago).

In addition to the immunolabeling of actin described above, F-actin
was also localized using fluorescent-labeled phalloidin. Fixed lens
sections were permeabilized in 0.2% Triton X-100 for 30 min, then
incubated with phalloidin conjugated to either fluorescein
isothiocyanate or tetramethyl rhodamine isothiocyanate (Sigma Chemical
Company) at a dilution of 1:100 of a methanolic stock (200 U/ml)
solution. Sections were washed 4 times in 0.07 M PBS for 10 min each,
then mounted and examined as detailed above.

Control experiments were conducted to ascertain whether the
fluorescence was specific binding or non-specific background. Lenses
were dissected and processed as previously described. However, normal
(non-immune) serum from the same species in which the primary antibody
was raised was utilized in place of the primary antibody as follows:
for myosin labeling, sections were treated with normal rabbit serum;
for ß-1 integrin and N-cadherin labeling, antibody was substituted with
normal goat serum; for pan-cadherin labeling, normal mouse serum was
used. Following incubation, the specimens were processed and viewed as
described above.

Detergent extraction

To assess whether the antigenic sites of some BMC components
(specifically cadherin and ß-1 integrin) were masked by changes in
molecular interactions during fiber elongation, some sections were
subjected to detergent extraction to ‘unmask’ antigens. Variations of
this technique have been successfully used in the lens to reveal
epitopes that were either inaccessible or masked during protein
rearrangements as a consequence of fiber differentiation [28,29]. In the present
study the method described by Beebe et al. [29] was utilized with minor modifications.
Specifically, fresh, unfixed lenses were mounted with cyanoacrylate
glue, embedded in 2% agar and sectioned with a sapphire knife at a
thickness of 300 µm. Sections were immediately placed in detergent
buffer (0.5% Nonidet P 40 Substitute, 100 mM KCl, 5 mM MgCl2,
1 mM EDTA disodium salt, 2 mM 2-mercaptoethanol, and protease inhibitor
cocktail) for 3 h at 4°C. Following extraction, sections were fixed and
immunolabeled as described above.

Data analysis

To locate and confirm the position of nascent suture branches,
paired confocal and differential interference contrast (DIC) images
were collected from most specimens. Our previous definitions of the
regions of fiber end migration were utilized for this assessment [27]. To summarize, the
4 regions of basal fiber end migration are: 1) equatorial, 2)
lateral-posterior (posterior from the equator to
within 150 µm of sutures), 3) peri-sutural (150 µm surrounding the
sutures), and 4) sutural. In order to thoroughly characterize BMC
components, both single-plane and z-series’ were collected at 25X, 40X,
and 60X magnification, at a pinhole of 1.0. Images were viewed and
analyzed using the Zeiss LSM Image Browser version 2.30.011 (Carl
Zeiss, Jena, Germany) and Adobe Photoshop version 7.0 (Adobe Systems
Inc, San Jose, CA).

To evaluate changes in labeling intensity, optical density line
scans were made across digital confocal images and plots were generated
using Scion Image v. beta 4.0.2 (Scion Corp., Frederick, MD). Line
scans were oriented 90º to suture branches and included both the
sutural and adjacent peri-sutural regions. Plots were inverted to show
high labeling signal as peaks and low labeling signal as troughs.

The distribution of selected BMC components was first established in
the equatorial region using whole-mount lens capsules (Figure 2). As
previously reported, [27]
F-actin (visualized with phalloidin-FITC) predominantly localized to
the periphery of the BMC with only faint actin fluorescence present
within the brighter profiles (Figure 2A,B). Labeling for myosin IIA
also showed strong peripheral fluorescence as well as definitive
labeling within the remainder of the BMC (Figure 2C,D). Cadherin distribution in
the equatorial region was assessed with a pan-cadherin antibody and
demonstrated the expected localization at lateral (cell-cell) borders
of the BMC (Figure
2E,F). Not surprisingly, cadherin immunofluorescence was not
detected at the basal membrane (where cells interface with the
capsule). Labeling for β-1 integrin in equatorial fibers was diffuse
and present as a basal plaque (Figure 2G; asterisks) as well as
prominent at lateral cell borders (Figure 2G,H).

A prior investigation of the BMC [1] utilized a modified decapsulation technique
(capsule-stripping) as well as examination of intact embryonic lenses
to assess components of the BMC. In order to establish the validity of
examining the BMC in Vibratome-sectioned material (i.e. absence of
preparative artifacts and structural disruption), a direct comparison
was made between fiber ends from decapsulated lenses and fiber ends in
posterior lens sections in the present study. This comparison also
provided important points of reference for interpretation of BMC
component-labeling in Vibratome-sectioned material. Specifically,
F-actin labeling was used to delineate fiber ends (in the posterior
lateral region), then visualized in through-focus z-series’. Each
z-series began at the capsule (z=0 µm) and proceeded inward through the
capsule-fiber interface (CFI), the fiber feet and (if present) the
lateral borders of underlying fibers. Results from the decapsulation
method (Figure 3A
and Animation 1) demonstrated that faint F-actin fluorescence began at
the CFI (z=1µm), gradually increasing to reveal distinct fiber end
profiles with diffuse staining in the remainder of the BMC. The lateral
borders of an underlying elongating fiber layer was observed by z=5µm;
presumably this cell layer adhered to the superficial fiber ends during
the prefixation step. In posterior sections (Figure 3B and Animation 2), actin
fluorescence was first detected at the CFI, with fiber end profiles
rapidly becoming visible (by z=1-2µm). Eventually, the lateral borders
of underlying fibers were visualized at z=5µm and were quite apparent
in subsequent optical sections. In both techniques, fiber feet had a
depth of approximately 4µm. Fiber end profiles in sectioned material
were slightly larger and more variable in size than those seen in the
decapsulation specimens, consistent with their more distal location in
the lateral-posterior region of fiber end migration [27].

Localization of BMC components in the peri-sutural and sutural
regions was examined in 100 µm vibratome sections of normal rat lenses.
Using the points of reference established in the above z-series
analysis, labeling within 2 µm of the CFI was considered to be due to
the BMC. As previously reported [27], F-actin labeling was predominantly
localized to the periphery of the BMC with faint actin fluorescence
present within the brighter profiles (Figure 4A-C). As the basal ends of the
lens fibers approached the posterior suture branches, the F-actin in
the BMC was rearranged into numerous smaller profiles (Figure 4C).
Additionally, labeling intensity appeared to be increased at sutures.
Optical density scans across sutural regions consistently showed an
increased level of fluorescence (Figure 4B; inset). Because prior
evidence indicated that actin was extensively associated with fodrin in
fiber end segments [30],
the distribution of fodrin was examined in the BMC. Double labeling for
F-actin and fodrin demonstrated a very high degree of colocalization in
the BMC; both in the sutural and peri-sutural regions (Figure 4D).

Labeling for myosin IIA showed abundant cytoplasmic fluorescence in
posterior portions of fiber cells (Figure 5A,C,F). In the peri-sutural
region, myosin was present in the BMC as a diffuse plaque, which filled
the fiber ends (Figure
5A; arrowheads). Myosin distribution in the BMC was not
altered in sutural regions (as compared to peri-sutural regions).
Specifically, double labeling for myosin and actin (Figure 5C-F)
showed fiber profiles with strong peripheral actin labeling, which were
filled with diffuse myosin label.

As expected, N-cadherin immuno-fluorescence was localized at the
periphery of the BMC in the peri-sutural region (Figure 6A,C).
Higher magnification revealed that labeling for cadherin was
distributed around the entire border of the BMC (Figure 6D;
inset). Additional experiments demonstrated the same marginal pattern
of N-cadherin labeling within the BMC in the lateral-posterior region
(data not shown). Double labeling for N-cadherin and F-actin
demonstrated that they were co-localized at the BMC border during fiber
end migration (Figure
6A-D). However, at and approaching the suture branches,
N-cadherin immuno-fluorescence appeared to be reduced (Figure 6A,C).
Because studies have demonstrated that several cadherins are expressed
in the developing lens [31,32], we utilized a
pan-cadherin antibody to assess the labeling distribution of cadherin
family proteins in the BMC of elongating fibers. The data showed the
same distribution around the periphery of the BMC in peri-sutural
regions and a clear decrease in cadherin labeling at basal fiber ends
in the sutural region (Figure
7A,B), thus confirming and extending the results obtained
for N-cadherin localization. Detergent extraction (to assess whether
the antigenic sites were masked) demonstrated a comparable labeling
pattern as in the tissue fixed and immunostained by standard
techniques. That is, there was a distinct lack of cadherin labeling at,
and directly adjacent to the sutural region (Figure 7C,D). In fully elongated fibers
that had detached from the capsule (Figure 7E,F), cadherin labeling was
lacking at the basal-to-basal fiber interface where fiber ends abut and
interdigitate to form the suture. However, strong cadherin labeling was
present along lateral aspects of posterior fiber segments flanking the
abutted fiber ends. Through focus analysis of the sutural region
revealed that fully-elongated fibers in the process of detaching from
the capsule and fibers already interdigitated at sutures lacked
cadherin in only the basal 3-4 µm.

As stated above, both antibodies against cadherin showed that it was
distributed around the entire periphery of the BMC. Because this data
contrasts with studies of cadherin distribution in cross-sections of
cortical lens fibers [33]
as well as in the chick lens BMC [1], equatorial segments of elongating fibers
were examined in the present study. Specifically, Vibratome sections
through the lens equator were obtained and double-labeled for actin and
cadherin (Figure 8).
The flattened hexagonal profiles of fiber cross-sections displayed
actin fluorescence concentrated along the short sides with faint label
along broad sides (Figure
8A). Similarly, cadherin labeling was more pronounced along
the short sides, while broad fiber faces demonstrated reduced and often
discontinuous label (Figure
8B; arrows). Merged actin and cadherin fluorescence
demonstrated far-reaching colocalization (Figure 8C).

To assess how several of the BMC components were distributed with
respect to one another, triple labeling with antibodies against actin,
pan-cadherin and myosin IIA was performed (Figure 9). The results were consistent
with the single and double labeling results presented above.
Specifically, in the peri-sutural region, both actin and cadherin
fluorescence delineated fiber end profiles, while myosin was
distributed diffusely throughout the fiber ends (as well as in the
cytoplasm of posterior fiber segments).

Immuno-labeling of β-1 integrin was carried out both separately and
with F-actin localization. In the peri-sutural region, localization of
β-1 integrin at the CFI showed that it was distributed throughout the
BMC as a diffuse plaque within the actin-rich borders (Figure 10).
Absent profiles of β-1 integrin within the fiber ends were also evident
(Figure 10C;
white asterisks). This may be attributed to preparative procedures that
may have inadvertently disrupted the adhesive properties of some of the
cells. Integrin labeling was much less prominent at the posterior
sutures as well as approaching the posterior sutures (Figure 11A,B).
Higher magnification of sutural regions revealed that β-1 integrin
labeling appeared to be markedly reduced as the lens fiber cells reach
the posterior sutures (Figure 11C,D). The detergent
extraction procedure had an unfortunate dampening effect on β-1
integrin labeling overall. However, sutural regions showed the same
reduction in β-1 integrin labeling as specimens subjected to standard
techniques (data not shown).

As negative controls, sections were treated with normal (non-immune)
serum from the same species in which the primary antibody was raised in
place of primary antiserum. In each case, specimens did not exhibit
specific labeling (data not shown), although a low level of background
was noted in some specimens.

The distribution of BMC components in normal rat lenses at the
initiation of fiber elongation/migration demonstrated both similarities
and differences with respect to that established in avian lenses in the
same general region [1].
Specifically, in both models, F-actin was abundant at lateral borders
of the BMC with faint actin fluorescence within the profiles. However,
sheared-off fiber ends from rat lenses did not display dendritic
processes around their periphery as seen in chicks. This suggests that
the attachment of nascent rat lens fibers to the capsule may be less
rigorous than that seen in avian lenses, since the dendrite-like
extensions are probably due to partial retraction of the cell borders
during the capsule-stripping technique. In addition, phalloidin-labeled
basal fiber ends in rats lacked the prominent foci of actin bundles
midway along hexagonal faces that were seen in embryonic chick lenses.
Similar to F-actin distribution, N-cadherin was present only at the
borders of the BMC in both models, but rat lenses showed a homogeneous
labeling intensity around the periphery of the BMC. In contrast,
N-cadherin was greatest at the midpoint of hexagonal faces in chick
lenses. In both types of lenses, myosin distribution was plaque-like;
however rat lenses also demonstrated strong peripheral BMC labeling
whereas in chick lenses myosin appeared to be concentrated toward the
center of each fiber end profile. Both models also demonstrated
positive labeling for β1 integrin, but a comparison of molecular
distribution in the BMC could not be made since the label was
visualized from different perspectives in each case.

In rat lenses, direct comparison of basal fiber ends either attached
to the capsule after decapsulation or within posterior sections,
demonstrated that the two techniques result in adequate and comparable
preservation of structure and BMC architecture. Thus, the BMC can be
reliably located and visualized in sectioned material without the
possibility of introducing mild artifacts during capsule stripping
(which creates tension on the capsule-fiber linkage). Through-focus
z-series analysis of intact basal fiber ends also revealed that fiber
feet are ‘boutons’ having a depth of approximately 4µm from the CFI to
the lateral borders of underlying fibers.

F-actin distribution in the BMC of rat lens fibers in more distal
locations was consistent with that seen in equatorial region and the
proximal portion of the posterior-lateral region (i.e. it was localized
principally to the periphery of basal fiber ends with dim fluorescence
within the remainder of the BMC). The predominantly peripheral
localization of actin in the BMC suggests that actin exerts its
greatest influence on cell migration at the borders of the fiber feet,
possibly controlling the dynamics of leading and trailing edges of the
basal membrane domain. This is consistent with evidence that various
regulatory components known to be involved in actin dynamics are also
present at the leading edge of migrating lens cells [34,35].

As fiber ends approached the sutural regions, the F-actin in the BMC
was rearranged into numerous smaller profiles and labeling for F-actin
was enhanced. As noted previously, the change in basal end morphology
is consistent with the idea that fiber ends round-up and partially
detach from the capsule as they approach the suture branch [27]. The increase in
labeling intensity could indicate that F-actin is somewhat enriched in
the BMC at the sutural regions. However, since the average size of
basal fiber ends in the sutural region is approximately 1/3 that of
fiber ends in the peri-sutural region [27], the increase in labeling intensity may, in
part, be due to the increased number of F-actin profiles per unit area.
The extensive colocalization of actin with fodrin in the BMC offers
another explanation for the persistent, strong actin fluorescence in
migrating basal fiber ends, despite the apparent paucity of adhesion
complexes at and approaching the sutures. Fodrin’s presence and
association with actin in the membrane skeleton of lens fibers has been
recognized for more than twenty years [36-38]
and more recent studies have shown that fodrin is present in both
elongating and maturing fibers [10,29,39]. Thus, the results
of the present investigation indicate that during the terminal stages
of elongation, actin is likely to be anchored in the BMC via its
interactions with fodrin and other membrane skeleton proteins.

N-cadherin, long known to be associated with lens fiber membranes [40,41] has since been
localized to lateral fiber membranes as well as the apical and basal
portions of elongating fibers [4,29,42]. It is therefore
not surprising that in the present study, N-cadherin fluorescence was
localized to the periphery of the BMC, coincident with the intersection
of the lateral and basal membrane domains, in most regions of fiber end
migration. Further, this data is somewhat consistent with results from
avian lenses [1],
and suggests a need for strong cell-cell adhesion during fiber end
migration. It is likely that a rigorous network of cell-cell adhesions
at the BMC periphery helps to maintain the established migration
patterns by allowing fibers to retain their relative positions adjacent
to fibers in the same growth shell and following those in the previous
growth shell. In fact, the requirement for cadherins in proper fiber
end migration is supported by a study of dexamethasone-treated lenses [43]. Specifically,
organ-cultured rat lenses developed PSCs in which the ordered
arrangement of elongating basal fiber ends was disrupted and a
concurrent decrease in cadherin expression was noted following
treatment.

Although cadherin was present in the BMC of chick and rat lenses,
its molecular distribution was not identical. Specifically, whereas
cadherin was distributed around the entire periphery of the BMC in rat
lenses, it was concentrated at the midpoints of each hexagonal face in
chick lenses [1].
This raises the question of whether cadherin distribution along the
anterior-posterior fiber length differs between species. Prior
investigations in bovine and chick lenses demonstrated that cortical
fiber mid-segments have a differential distribution of both cadherin
and actin to the short faces of flattened hexagonal cross-sections [1,33]. This is
consistent with the results of the present investigation, which
demonstrate a comparable labeling pattern in the mid-segments of
elongating rat lens fibers (Figure 8). It appears that in chick and
rat lenses the distribution of cadherin and actin differs in lateral as
opposed to basal membranes. This underscores the idea that the BMC
defines a distinct membrane domain.

In the present study, N-cadherin fluorescence in the BMC was
markedly reduced approaching and within the sutural region of fiber end
migration. This finding is somewhat similar to a previous investigation
that showed decreased N-cadherin labeling on lateral fiber membranes
soon after fibers detached from the capsule [29]. E-, N-, and B-cadherins have all been
detected during lens development, however, E-cadherin is only present
in lens epithelial cells [31,32]. Labeling of
elongating fibers using a pan-cadherin antibody demonstrated a lack of
cadherin immunofluorescence at nascent sutures in the current study.
This labeling distribution persisted even after detergent extraction to
uncover antigens that may have been masked by changes in molecular
arrangements during the terminal stages in elongation and fiber end
migration. These data indicate that the classical cadherins do not
appear to be localized to the basal domains during fiber end detachment
from the capsule and interdigitation to form sutures. However, the fact
that cadherins (and presumably adhering junctions) were still present
in the lateral membranes directly adjacent to the BMC suggests that
cadherin may simply be rearranged within fiber posterior segments
rather than lost. Such an arrangement would provide the necessary
cell-cell contacts to maintain fiber organization both between and
within growth shells.

This is the first investigation to directly show that sutures lack
the appropriate adhesion molecules for adhering junctions. However,
this is not surprising since, to date, the evidence supports the idea
that no junctions cross the sutures. In fact, the absence of any
discernable ‘junctional apparatus’ at sutures was first noted by
Kuwabara in his classic study of lens ultrastructure [44]. Subsequent
reports demonstrated that fibers are not connected across sutures by
gap junctions [45]
and that MIP is extremely sparse at apical tips of elongating fibers [46] as well as absent
from their basal domains [1],
making it a poor candidate for adhesion across sutures. Other common
junctions, including tight junctions, desmosomes and hemidesmosomes are
absent from lens fibers altogether [47-50].
The basis for adhesion of fiber ends at sutures remains unknown and may
simply be comprised of structural interdigitations that provide only
limited linkage between opposing groups of fibers.

In the peri-sutural region, β1 integrin was localized within the
actin-rich borders of fiber ends and superimposed over the weaker
F-actin fluorescence throughout the BMC. The data is consistent with
previous findings that actin filaments are linked to integrins that
connect the basal domain of the fiber cells to the overlying elastic
lens capsule [3].
The plaque-like appearance of integrin labeling in the BMC probably
reflects the arrangement of these receptors throughout the BMC, where
they participate in focal contacts between the cell and matrix
components in the capsule [51-55].

At and directly adjacent to the posterior sutures, labeling for β1
integrin was markedly decreased. These results contrast with a prior
study that showed robust immunoreactivity for β1 integrin near the
posterior pole in embryonic rat lenses [56]. The observed differences in integrin
distribution may be due to temporal differences in protein distribution
as a consequence of developmental stage. In the present study,
detergent extraction repeatedly resulted in an overall decrease in β1
integrin labeling, however, sutural domains remained virtually empty of
label, consistent with the labeling patterns noted in fixed,
unextracted lens slices. While somewhat difficult to interpret, these
results suggest that there may be a reduction in the overall number or
type of integrin receptors that are associated with the capsule as
fiber ends complete their migration and prepare to detach. One possible
explanation for the decreased labeling is down regulation of integrin
expression during the terminal phases of fiber end migration. In fact,
studies have demonstrated that expression of some β1 integrins (α6β1B
and α3β1) are down regulated during differentiation in avian lenses [2,50]. Although these
changes take place early in differentiation of lens fibers, an
analogous down regulation may take place during the terminal stages of
fiber elongation in rats, and could underlie the change in labeling
distribution in the present study. Specifically, down regulation of one
sub-type of integrin receptor could result in a change in the
predominant integrin type thereby creating a less adhesive attraction
to the lens capsule in preparation for timely fiber end detachment.

The results of this investigation indicate that the distribution of
some BMC components alters as fiber ends approach their sutural
destinations. Specifically, F-actin, the cadherins and β1 integrin all
undergo rearrangement to some degree during the final stage of fiber
end migration. These changes are illustrated in a summary diagram (Figure 12).
Furthermore, redistribution of BMC components appears to occur in a
defined, sequential fashion, which probably facilitates the terminal
migration and detachment of fibers from the capsule in order to form
orderly sutures. This is significant because disruption of these
processes would be likely to result in faulty migration leading to
sutural malformations, which affect lens function [17,18] and in extreme
cases may result in posterior subcapsular cataract formation [21,26].

The marked differences in basal fiber end morphology as well as BMC
architecture between lenses having branched and branchless suture
patterns have direct correlates in fully elongated fibers, in that they
impact lens shape, fiber organization, and the overall sutural anatomy
of each model system. Specifically, both fiber end structure and BMC
architecture in chick lenses appear more organized than in rat lenses.
For example, basal fiber ends in rat lenses vary significantly in size
and shape as they migrate across the capsule, having the most uniform
shape and basal end area just posterior to the equatorial region.
However, as fiber ends progress toward their sutural destinations,
uniformity is rapidly lost [27].
This contrasts with fiber ends in chick lenses, which are more regular
in size and arranged in ordered rows [1,49,57]. The regular array
of hexagonal fiber ends in chick facilitates the alignment of BMC
components into a lattice-like arrangement that imparts contractile
tone and may affect the radius of curvature of the posterior surface
and even assist in accommodation [1]. Conversely, the relative disorder of basal
fiber ends in the rat precludes the formation of a highly-organized
molecular lattice in the BMC. This is consistent with the facts that in
the rat eye, the posterior lens curvature normally remains constant [58] and active
accommodation is effectively zero [59]. The superior organization of avian BMC is
probably also correlated to the more rigorous adhesion of basal ends to
the capsule as compared to rat. A logical corollary, since the lack of
accommodation in rat lenses obviates the need to withstand the forces
that would be exerted at the capsule-fiber interface during this
process.

The specific migration pattern of fiber ends in a given system is a
crucial factor in producing a particular sutural configuration that, in
turn, affects the optical properties and focal abilities of the lens.
For example, in avian lenses, all migrating fiber ends maintain their
orientation directly toward the posterior pole [49,60], resulting in
fibers that approximate meridians and an umbilical or branchless
suture. In the rat, fibers initially migrate along meridians until they
reach a latitudinal ring defined by the proximal extent of the suture
branches [27].
Whereas the fiber ends of straight fibers that elongate to the proximal
ends of sutures have completed their migration, the fiber ends of
straight fibers that will eventually elongate to the distal end of
suture branches (at the pole) continue to migrate along meridians. All
other fiber ends migrate along paths that diverge from meridians after
passing within the latitudinal ring such that they begin to migrate
along a curved path, eventually interfacing with opposing fiber ends
and aligning as longitudinal arc lengths. Thus, the variable migration
paths of fiber ends within each growth shell results in opposite end
curvature (S-shaped fibers) and a branched suture pattern.

In avian lenses, the contraction of the ciliary body muscles pushes
the ciliary processes against the lens, compressing it into a
more-rounded overall shape [61].
At the fiber cell level, the pressure of the ciliary processes forces
the meridian-like fibers together at the poles, where their
highly-tapered ends overlap and produce a large change in the surface
curvature (lenticonus) [62].
Thus the broad accommodative range is a direct result of fiber shape
and sutural anatomy (which derives from the migration patterns of
elongating fiber ends). In contrast, mammalian lenses have S-shaped
fibers [60]. The
S-shaped fibers in primates resemble and function as simple springs,
which when expanded, results in the overlap of flattened fiber ends at
sutures [63].
Although the migration patterns of lenses with Y (and line) sutures
result in S-shaped, simple spring fibers, they lack sufficient end
taper to facilitate overlap at suture branches [62].

Although comparatively little data has, as yet, been collected on
the structure and molecular components that characterize the apical
fiber ends migrating across the lens epithelium, it can be assumed that
at least some of the same mechanisms are involved in the organization
and regulation of this process. Further study into the factors
directing fiber end migration at both apical and basal fiber ends is
clearly warranted.